Poly(ADP-ribosylation) and genomic stability.

Poly(ADP-ribose) polymerases (PARPs) catalyze the synthesis of ADP-ribose polymers and attach them to specific target proteins. To date, 6 members of this protein family in humans have been characterized. The best-known PARP, PARP-1, is located within the nucleus and has a major function in DNA repair but also in the execution of cell death pathways. Other PARP enzymes appear to carry out highly specific functions. Most prominently, the tankyrases modify telomere-binding proteins and thereby regulate telomere maintenance. Since only a single enzyme, poly(ADP-ribose) glycohydrolase (PARG), has been identified, which degrades poly(ADP-ribose), it is expected that this protein has important roles in PARP-mediated regulatory processes. This review summarizes recent observations indicating that poly(ADP-ribosylation) represents a major mechanism to regulate genomic stability both when DNA is damaged by exogenous agents and during cell division.     

Bipartite structure of the downstream element of the mouse beta globin (major) poly(A) signal.

The downstream region of the mouse beta (major) globin poly(A) signal was mutated and analyzed for function in transfected COS cells. From analysis of unidirectional Bal31 deletions, the 3' boundary of the downstream element was defined as +22 (22 nucleotides downstream from the cleavage site). Analysis of cluster mutations, in which 5 or 6 adjacent bases were replaced with a random CA-containing sequence in a manner that did not alter spacing, confirmed +22 as the 3' boundary of the downstream element. The analysis also revealed two short UG-rich sequences, located from +5 to +10 and from +17 to +22, as major functional components. In contrast, a more refined series of mutations, in which clusters of 3 bases were replaced, failed to cause loss of function. We conclude that the downstream element of the mouse beta globin poly(A) signal is bipartite in structure, and that portions of its sequence are functionally redundant.     

Mutations in poly(A) site downstream elements affect in vitro cleavage activity.

Previous studies have shown that a sequence element downstream of the poly(A) addition site is required for efficient cleavage in vivo. We tested a group of downstream element point mutations in an in vitro reaction using HeLa cell nuclear extract as a source of cleavage activity. In close agreement with earlier studies (M. A. McDevitt, R. P. Hart, W. W. Wong, and J. R. Nevins, EMBO J. 5:2907-2913, 1986), a downstream element from the adenovirus E2a gene directed a higher level of cleavage activity than one from the simian virus 40 early gene. Furthermore, a single-base change in the downstream element could result in a decrease in cleavage activity of about 50-fold. That these mutations have similar effects in vivo and in vitro indicates that the HeLa cell nuclear extract system contains all of the factors required to study the mechanism of sequence recognition.     

Poly(A), poly(A) binding protein and the regulation of mRNA stability

This review has focused on the possibility that interactions between mRNA sequences and the poly(A)-nucleoprotein complex play important roles in mRNA turnover. It is important to stress that additional genetic and biochemical tests are necessary to characterize how PABP interacts with mRNA in cells and to determine whether the poly(A) protection hypothesis is accurate. Moreover, there may be a significant number of mRNAs whose half-lives are independent of polyadenylation. For example, the stabilities of poly(A)-containing and deadenylated alpha 2u-globulin and interferon mRNAs are similar in microinjected oocytes. Thus, an important challenge in this field will be to analyse the complex and interactive factors that determine the half-lives of specific mRNAs.     

The site of 3'' end formation of histone messenger RNA is a fixed distance from the downstream element recognized by the U7 snRNP.

Two conserved elements direct the 3' end processing of histone messenger RNA: a stem-loop structure immediately upstream of the site of cleavage and the histone downstream element (HDE), located 12-19 nucleotides downstream of the stem-loop in the premessenger RNA. We studied the role of these two elements by systematically inserting up to 10 C residues between them in the mouse H2A-614 histone pre-mRNA. 3' End mapping of RNAs processed in vitro demonstrated that as the HDE is move downstream, the site of cleavage correspondingly moves 3'. In addition, the efficiency of processing declines. In the wild-type substrate, cleavage occurs 3' of an A residue; modest increases in the efficiency of processing of the insertion mutants were observed when an A residue was placed at the new cleavage site. The results of psoralen cross-linking studies and immunoprecipitations using anti-trimethylguanosine antibodies indicated that the decreased processing efficiency of the insertion mutants is not due to impaired binding of the U7 small nuclear ribonucleoprotein (snRNP). We conclude that the mammalian U7 snRNP acts as a molecular ruler, targeting enzymatic components of cleave histone pre-mRNAs a fixed distance from its binding site, the HDE.     

The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU)

Poly(dA.dU), which is specifically tritiated at the 1'-, 2'- (ribo configuration), 3'-, or 4'-position of deoxyuridine, has been synthesized and the fate of the tritium has been determined upon degradation of the polymer by bleomycin, Fe(II), and O2. No tritium is labilized from the 1'-3H-labeled polymer as 3H2O; however, the resulting 3-(uridin-1'-yl)-2-propenal (uracil propenal) has the expected specific activity. The 2'-3H-labeled polymer affords 3H2O and no label in the uracil propenal. This result and the lack of solvent incorporation into the uracil propenal suggest that proton abstraction from C-2' to afford the trans-propenal is highly stereospecific. For the 3'-3H-labeled polymer, 3H2O is formed and the specific activity of the uracil propenal is identical to that of the deoxyuridine. This suggests that the labilization of the 3'-H is exclusively associated with free uracil formation. 3H2O is also formed from the 4'-3H-labeled polymer. These findings along with previous studies are consistent with the formation of uracil propenal and free uracil by the trapping of the initially formed 4'-radical species by O2 or by a monooxygen species, respectively.     

Commitment to splice site pairing coincides with A complex formation

Differential recognition of exons by the spliceosome regulates gene expression and exponentially increases the complexity of metazoan proteomes. After definition of the exons, the spliceosome is activated by a series of sequential structural rearrangements. Formation of the first ATP-independent spliceosomal complex commits the pre-mRNA to the general splicing pathway. However, the time at which a commitment to a specific splice site choice and pairing is made is unknown. Here, we demonstrate that alternative splicing patterns are irreversibly chosen at a kinetic step different from the ATP-independent commitment to splicing. Splice sites become committed at the first ATP-dependent spliceosomal complex when rearrangements lock U2 snRNP onto the pre-mRNA. Thus, commitment to the splicing pathway and commitment to splice site pairing are separate steps during spliceosomal assembly, and ATP hydrolysis drives the irreversible juxtaposition of exons within the spliceosome.     

The downstream regulatory element of the proU operon of Salmonella typhimurium inhibits open complex formation by RNA polymerase at a distance

The intracellular concentration of K(+)-glutamate, chromatin-associated proteins, and a downstream regulatory element (DRE) overlapping with the coding sequence, have been implicated in the regulation of the proU operon of Salmonella typhimurium. The basal expression of the proU operon is low, but it is rapidly induced when the bacteria are grown in media of high osmolarity (e.g. 0.3 M NaCl). It has previously been suggested that increased intracellular concentrations of K(+)-glutamate activate the proU promoter in response to increased extracellular osmolarity. We show here that the activation of the proU promoter by K(+)-glutamate in vitro is nonspecific, and the in vivo regulation cannot simply be mimicked in vitro. In vivo specificity requires both the chromatin-associated protein H-NS and the DRE; they are both needed to maintain repression of proU expression at low osmolarity. How H-NS and the DRE repress the proU promoter in vivo has so far been unclear. We show that, in vivo, the DRE acts at a distance to inhibit open complex formation at the proU promoter.